Schultz N M, Huang L, Kennedy R T
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Anal Chem. 1995 Mar 1;67(5):924-9. doi: 10.1021/ac00101a020.
The development of a competitive immunoassay for insulin that is based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) is described. The assay uses fluorescein isothiocyanate-derivatized insulin (FITC-insulin) as a tracer and the Fab fragment of an anti-insulin monoclonal antibody as the immunoreagent. Reproducibility and reliability of the assay are improved when CZE capillaries are coated with a neutral hydrophilic polymer. The assay has an average relative standard deviation of 3.4% and a detection limit of 3 nM or about 6 fg injected. Using the assay, insulin content of single islets of Langerhans was determined to be 35 +/- 4 ng, which is in good agreement with literature values. The assay was also used to determine insulin secretion from single islets. During 5-min static incubations, 3 mM glucose elicited 0.8 +/- 0.2 ng of insulin release while 16 mM glucose elicited 3.2 +/- 0.5 ng of insulin release. These values are also in good agreement with previously reported results. These applications demonstrate the capability of rapidly determining a low level of protein in a complex biological microenvironment.
本文描述了一种基于毛细管区带电泳-激光诱导荧光检测（CZE-LIF）的胰岛素竞争性免疫分析方法的开发。该分析方法使用异硫氰酸荧光素衍生化的胰岛素（FITC-胰岛素）作为示踪剂，抗胰岛素单克隆抗体的Fab片段作为免疫试剂。当CZE毛细管涂覆中性亲水性聚合物时，该分析方法的重现性和可靠性得到提高。该分析方法的平均相对标准偏差为3.4%，检测限为3 nM或约6 fg进样量。使用该分析方法，测定单个胰岛的胰岛素含量为35±4 ng，与文献值吻合良好。该分析方法还用于测定单个胰岛的胰岛素分泌。在5分钟的静态孵育过程中，3 mM葡萄糖引发0.8±0.2 ng胰岛素释放，而16 mM葡萄糖引发3.2±0.5 ng胰岛素释放。这些值也与先前报道的结果吻合良好。这些应用证明了在复杂生物微环境中快速测定低水平蛋白质的能力。
